Ultrafast Non-Förster Energy Transfer in a DNA-Templated Cy3-Cy5 Assembly

Controlling electronic coupling between chromophores is central to the design of photonic materials, yet energy transfer at sub nanometer donor-acceptor separations, where the point-dipole Förster approximation becomes unreliable, remains difficult to probe experimentally. Here, we use a doublestranded DNA scaffold to position a Cy3 donor and a Cy5 acceptor within a few ångströms of each other and characterize their energy transfer using steady-state and femtosecond transient absorption spectroscopy, microsecond molecular dynamics and umbrella-sampling simulations, and TDDFT-based transition-charge (TrESP) coupling calculations. Absorption changes consistent with H-type heterodimer formation accompany a near-unity (0.96) energy-transfer efficiency, while Cy3 signal depletes and Cy5 signal rises within the ~200 fs instrument response. Simulations show that the Cy3 and Cy5 are moderately-to-strongly coupled in the vast majority of the thermal ensemble, leading to energy transfer lifetimes shorter than 150 fs, consistent with transient absorption measurements. These results establish DNA scaffolds as programmable platforms for accessing non-Förster energy-transfer regimes.